In the medical and pharmaceutical fields, as well as in other areas, such as nutrition, etc., it is often necessary to determine the toxicity of certain gaseous, liquid and/or viscous substances.
The conventional processes at the present time call first of all, for cell culture tests. Subsequently animal tests are added. This is especially the case when in the cell culture tests, no toxicity has been ascertained. Then, if necessary, clinical studies on humans are undertaken. In particular, the prior art can be described as follows:
Model systems currently used to determine acute toxicity of liquid or particular substances are based on the use of cell cultures. Adherently growing cells and non-adherently growing cells are exposed for different time intervals to the substance to be tested, which is either offered with the nutrient solution or is to be colonized as a culture substrate by the cells.
Application of gaseous or liquid test substances is done in this system jointly with the test substance. Specific polar application of the test substance to the basal or apical aspect of epithelial cell monolayer is not done. The cells are either completely flushed by the medium and test substance, or exposed to both components jointly in the area of the apical cell membrane.
It is characteristic of these systems that they work only with a certain cell type. Often they are cells capable of unlimited division, such as cells with tumor characteristics.
Both epithelial cell lines and also cell lines with connective tissue characteristics are used for these tests. They differ in their properties however significantly from differentiated epithelial or connective tissue cells in human or animal organs, since tumor cells are dedifferentiated and can execute the typical cellular functions of differentiated cells only to a limited degree. According to the knowledge underlying the invention, this is one of the reasons for the lack of applicability of results obtained in cell culture experiments to the situation in the organism. Chronic toxic effects on cells are unsatisfactorily studied using cell culture systems.
If test substances in the cell culture systems have been found to be nontoxic, they must be studied for their action in an animal organism in different ways. Still, results of animal tests can only be applied to humans to a limited degree. There are major differences in the metabolic event between the human and animal organism. On the one hand, substances toxic to certain species are tolerated by humans without extensive adverse side effects (example: aspirin). On the other hand, there are a large number of test substances which are recognized as toxic to humans only in clinical studies.
One disadvantage of animal tests is that they are timeconsuming and often indicate little with reference to the human organism. Likewise, clinical tests on humans are expensive and complex and often cannot be done.
In particular, a process is disclosed for the in-vitro testing of effects on biological structures in German patent No. DE 42 29 013 A1. The process is an analytic technique, and a tolerance, toxicity, side effect and efficacy test. In this process, the respective sample is placed in a chamber in which it is completely flushed by the test substance so that disadvantages arise. These are the same disadvantages that were indicated above in the discussion of cell culture tests.
Further, a system or process is known for measuring transepithelial resistances on tissue samples with a small diameter as disclosed in German Patent No. DE 40 20 013 A1. The patent uses a perfusion chamber for holding the respective tissue sample. In this system, small organisms or organs are studied with respect to their transport properties (transport systems of the membrane for ions) with special application areas of ecotoxicology, pharmacology and transport physiology in biology and medicine. The use of this known process is limited exclusively to epithelial cells. Transepithelial resistance is determined.
Further, a device is known for treatment of tissue samples held in vitro and drawn up on glass slides as described in German Patent No. DE 36 35 013 C2. The device has a chamber for holding the preparation. This chamber can be filled with the respective treatment reagent, can be sealed tight with a cover and can be provided with feed and drain openings for the reagents. It is basically a single-chamber system. The respective sample itself is located on the impermeable glass slide and is wetted on only one side by the respective test liquid. In this system, cells of the sample can only execute typical cellular functions of differentiated cells to a very limited degree, or not at all. The results obtained are not applicable or only conditionally applicable or only conditionally applicable to the actual situation in a specific organism.
Further, a device is known for determining and measuring membrane-permeable substances (see U.S. Pat. No. 5,525,475). This device does not determine tolerance, for example, the toxicity of gaseous, liquid or viscous substances for the human or animal mechanism.
The device calls for two chambers which are separated from one another by a semipermeable membrane. The substance to be tested is placed in one of the chambers. The diffusion of the substance is measured in a second chamber. Cultivation of cells or tissues is not provided in either of these chambers. The purpose of this system is the determination of the diffusibility of a test substance, not the determination of its biological action, especially not the determination of tolerance for human or animal organisms.
An object of the invention is to devise a process which avoids these defects and enables the determination of the tolerance or toxicity of gaseous, liquid or viscous substances in the laboratory for a human or animal mechanism.